Prokaryotic Expression, Purification, and Polyclonal Antibody Production of a Truncated Recombinant Rabies Virus L Protein

Zhang, Jinyang; Jin, Zian; Sun, Tung‐Tien; Jiang, Yan; Han, Qinqin; Song, Yuzhu; Chen, Qiang; Xia, Xin

doi:10.15171/ijb.1022

Cited by 7 publications

(8 citation statements)

References 37 publications

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“…RABV L protein was also reported as the potential target for diagnosis [ 3 ]. Here, we immunized mice with purified truncated L protein (aa 1429–1757) of CVS-11, which was reported to efficiently induce the antibody response in mice [ 13 ], and generated three mAbs against L protein, designated as 3F3, 3A6 and L-C. All of the mAbs could recognize the RABV L protein of CVS-11 by IFA and western blot, showing good reactivity for RABV ( Figure 2 D). RABV is an ancient virus and causes an incurable disease, thus the detection and monitoring of rabies virus (RABV) is still very important.…”

Section: Discussionmentioning

confidence: 99%

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Development of Monoclonal Antibodies for Detection of Conserved and Variable Epitopes of Large Protein of Rabies Virus

Zhao

et al. 2021

Viruses

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Rabies virus (RABV) causes fatal neurological encephalitis and results in approximately 6000 human death cases worldwide every year. The large (L) protein of RABV, possessing conserved domains, is considered as the target for detection. In this study, three monoclonal antibodies (mAbs), designated as 3F3, 3A6 and L-C, against L protein were generated by using the recombinant truncated L protein (aa 1431–1754) and the epitopes were also identified using a series of overlapping truncated polypeptides for testing the reactivity of mAbs with different RABV strains. The 1479EIFSIP1484, 1659RALSK1663 and 1724VFNSL1728 were identified as the minimal linear epitopes recognized by mAbs 3F3, 3A6 and L-C, respectively. Amino acid alignment showed epitope 1724VFNSL1728 recognized by mAb L-C is completely conserved among RABV strains, indicating that mAb L-C could be used to detect all of the RABV strains. Epitope 1479EIFSIP1484 is highly conserved among RABV strains except for a P1484S substitution in a China I sub-lineage strain of Asian lineage, which eliminated the reactivity of the epitope with mAb 3F3. However, the epitope 1659RALSK1663 was only completely conserved in the Africa-2 and Indian lineages, and a single A1660T substitution, mainly appeared in strains of the China I belonging to Asian lineage and a Cosmopolitan lineage strain, still retained the reactivity of the epitope with mAb 3A6. While both A1660T and K1663R substitutions in a China I lineage strain, single K1663R/Q substitution in some China II strains of Asian lineage and some Arctic-like lineage strains and R1659Q mutation in a strain of Africa-3 lineage eliminated the reactivity of the epitope with mAb 3A6, suggesting mAb 3A6 could be used for differentiation of variable epitopes of some strains in different lineages. Thus, variability and conservation of the three epitopes of L protein showed the reactive difference of mAbs among RABV strains of different lineages. These results may facilitate future studies in development of detection methods for RABV infection, the structure and function of RABV L protein.

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Section: Discussionmentioning

confidence: 99%

“…The truncated L protein (aa 1429–1757) of RABV in prokaryotic expression system could induce the antibody response efficiently by immunizing mice, suggesting that this fragment had good antigenicity [ 13 ]. Thus, we select the fragment as antigen to prepare mAbs against L protein.…”

Section: Methodsmentioning

confidence: 99%

Development of Monoclonal Antibodies for Detection of Conserved and Variable Epitopes of Large Protein of Rabies Virus

Zhao

et al. 2021

Viruses

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“…LDH isoenzymes stocks were finally stored in PBS (pH = 8.0) at 4 °C until following steps. The LDH isoenzymes were prepared simultaneously under the same conditions [ 60 ].…”

Section: Methodsmentioning

confidence: 99%

Enzymatic Kinetic Properties of the Lactate Dehydrogenase Isoenzyme C4 of the Plateau Pika (Ochotona curzoniae)

Wang¹,

Lian²,

Wei³

et al. 2016

IJMS

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Testis-specific lactate dehydrogenase (LDH-C4) is one of the lactate dehydrogenase (LDH) isozymes that catalyze the terminal reaction of pyruvate to lactate in the glycolytic pathway. LDH-C4 in mammals was previously thought to be expressed only in spermatozoa and testis and not in other tissues. Plateau pika (Ochotona curzoniae) belongs to the genus Ochotona of the Ochotonidea family. It is a hypoxia-tolerant species living in remote mountain areas at altitudes of 3000–5000 m above sea level on the Qinghai-Tibet Plateau. Surprisingly, Ldh-c is expressed not only in its testis and sperm, but also in somatic tissues of plateau pika. To shed light on the function of LDH-C4 in somatic cells, Ldh-a, Ldh-b, and Ldh-c of plateau pika were subcloned into bacterial expression vectors. The pure enzymes of Lactate Dehydrogenase A4 (LDH-A4), Lactate Dehydrogenase B4 (LDH-B4), and LDH-C4 were prepared by a series of expression and purification processes, and the three enzymes were identified by the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (PAGE). The enzymatic kinetics properties of these enzymes were studied by Lineweaver-Burk double-reciprocal plots. The results showed the Michaelis constant (Km) of LDH-C4 for pyruvate and lactate was 0.052 and 4.934 mmol/L, respectively, with an approximate 90 times higher affinity of LDH-C4 for pyruvate than for lactate. At relatively high concentrations of lactate, the inhibition constant (Ki) of the LDH isoenzymes varied: LDH-A4 (Ki = 26.900 mmol/L), LDH-B4 (Ki = 23.800 mmol/L), and LDH-C4 (Ki = 65.500 mmol/L). These data suggest that inhibition of lactate by LDH-A4 and LDH-B4 were stronger than LDH-C4. In light of the enzymatic kinetics properties, we suggest that the plateau pika can reduce reliance on oxygen supply and enhance its adaptation to the hypoxic environments due to increased anaerobic glycolysis by LDH-C4.

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“…The proteins loaded (40 mg per lane) were subsequently separated by 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (PVDF; Thermo Fisher Scientific, Inc.), blocked at 37˚C for 2 h in 5% non-fat milk, and incubated with anti-proliferating cell nuclear antigen (PCNA; sc-25280) or anti-ISCU antibody (sc-271536) or β-actin antibody (sc-47778) or GAPDH (sc-25778) at 1:4,000 or 1:3,000 or 1:10,000 or 1:5,000, respectively (Santa Cruz Biotechnology, Inc.) at 4˚C overnight. After washing for 10 min in 1X Tris-buffered saline-Tween-20 solution three times, the target protein was probed with the horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (sc-3841; at 1:6,000 dilution; Santa Cruz Biotechnology, Inc.) at 37˚C for 2 h. After washing for 10 min in 1X Tris-buffered saline-Tween-20 solution three times, the bound antibody on the PVDF membrane was detected by chemiluminescence with the ECL Detection Reagent kit (Pierce; Thermo Fisher Scientific, Inc.) and the protein expression was analyzed using Quantity One 4.6.2 (Bio-Rad Laboratories, Inc.) (16).…”

Section: Rna Transfection a Total Of 2x10mentioning

confidence: 99%

MitoKATP channels promote the proliferation of hypoxic human pulmonary artery smooth muscle cells via the ROS/HIF/miR‑210/ISCU signaling pathway

Ding

Wang

et al. 2017

Exp Ther Med

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Abstract.Previous results have indicated that mitochondrial ATP-sensitive potassium (mitoK ATP ) channels are associated with the hypoxic proliferation of pulmonary artery smooth muscle cells (PASMCs). However, the mechanism underlying the promotive effects of mitoK ATP channels on cell proliferation in response to hypoxia remains unknown. mitoK ATP channel opening results in a collapse of mitochondrial membrane potential and generation of mitochondrial reactive oxygen species (ROS). As hypoxia-inducible factor-1α (HIF-1α) is a critical oxygen sensor and major transcriptional regulator of the hypoxic adaptive response, the current study assessed whether mitoK ATP opening contributes to the chronic proliferation of human PASMCs (hPASMCs) in collaboration with HIF-1α and its downstream targets under hypoxic conditions. The present study demonstrated that there was crosstalk between mitoK ATP channels and HIF-1α signaling in PASMCs under hypoxic conditions. The results suggest that mitoK ATP channels are involved in the proliferation of PASMCs during hypoxia through upregulation of the ROS/HIF/microRNA-210/iron-sulfur cluster protein signaling pathway. IntroductionPulmonary arterial hypertension (PAH) is a life-threatening disorder characterized by obstructive remodeling of the pulmonary arteries, which may lead to right-sided heart failure and mortality (1-2). PAH may be defined as a mean pulmonary artery pressure (mPAP) of ≥20 mmHg at rest or >30 mmHg with exercise, along with a pulmonary artery occlusion pressure of ≤15 mmHg (1-2). The estimated incidence of primary pulmonary hypertension is 1-2/million in the global population (1-2). There are currently three treatment pathways, namely phosphodiesterase type 5 inhibitor or soluble guanylate cyclase stimulator, prostacyclin class therapy and endothelium receptor antagonist, which target the imbalances of three substances; nitric oxide, prostacyclin and endothelin, respectively (3). Despite the efficacy of these pharmacological therapies in improving symptoms, they do not prevent the progression of PAH or reduce the mortality rate of patients (3). Therefore, novel approaches that more effectively target PAH are required to control the cellular components associated with pulmonary remodeling. The chronic and continued proliferation of human pulmonary artery smooth muscle cells (hPASMCs), in addition to apoptotic resistance, leads to hypoxic pulmonary arterial remodeling, which is considered to be a key mechanism for the pathological development of PAH (4). ATP-sensitive potassium (K ATP ) channels are located on the cytoplasmic membrane and on subcellular membranes. Subcellular membrane-associated K ATP channels are divided into three types: Sarcolemmal K ATP , mitochondrial K ATP (mitoK ATP ) and nuclear K ATP . MitoK ATP channels, which contribute to the control of mitochondrial volume and energetic status (5-7), exhibit high sensitivity to hypoxia (8

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Prokaryotic Expression, Purification, and Polyclonal Antibody Production of a Truncated Recombinant Rabies Virus L Protein

Cited by 7 publications

References 37 publications

Development of Monoclonal Antibodies for Detection of Conserved and Variable Epitopes of Large Protein of Rabies Virus

Development of Monoclonal Antibodies for Detection of Conserved and Variable Epitopes of Large Protein of Rabies Virus

Enzymatic Kinetic Properties of the Lactate Dehydrogenase Isoenzyme C4 of the Plateau Pika (Ochotona curzoniae)

MitoKATP channels promote the proliferation of hypoxic human pulmonary artery smooth muscle cells via the ROS/HIF/miR‑210/ISCU signaling pathway

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